Accepted Manuscript 4β-[4’-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA Topoisomerase I and IIα Inhibitors and Apoptosis Inducing Agents Ahmed Kamal, Paidakula Suresh, M. Janaki Ramaiah, T. Srinivasa Reddy, Ravi Kumar Kapavarapu, Bolla Narasimha Rao, Syed Imthiajali, T. Lakshminarayan Reddy, S.N.C.V.L. Pushpavalli, Nagula Shankaraiah, Manika Pal-Bhadra PII: S0968-0896(13)00564-6 DOI: http://dx.doi.org/10.1016/j.bmc.2013.06.033 Reference: BMC 10926 To appear in: Bioorganic & Medicinal Chemistry Received Date: 26 April 2013 Revised Date: 12 June 2013 Accepted Date: 13 June 2013 Please cite this article as: Kamal, A., Suresh, P., Janaki Ramaiah, M., Srinivasa Reddy, T., Kapavarapu, R.K., Rao, B.N., Imthiajali, S., Lakshminarayan Reddy, T., Pushpavalli, S.N.C.V.L., Shankaraiah, N., Pal-Bhadra, M., 4β- [4’-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA Topoisomerase I and IIα Inhibitors and Apoptosis Inducing Agents, Bioorganic & Medicinal Chemistry (2013), doi: http://dx.doi.org/10.1016/j.bmc.2013.06.033 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Accepted Manuscript
4β-[4’-(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA Topoisomerase I
and IIα Inhibitors and Apoptosis Inducing Agents
Ahmed Kamal, Paidakula Suresh, M. Janaki Ramaiah, T. Srinivasa Reddy, Ravi
Kumar Kapavarapu, Bolla Narasimha Rao, Syed Imthiajali, T. Lakshminarayan
4 -[4 -(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA Topoisomerase I and IIα Inhibitors and Apoptosis Inducing Agents
Ahmed Kamal,*a Paidakula Suresh,a M. Janaki Ramaiah,b T. Srinivasa Reddy,a,c Ravi Kumar Kapavarapu,d Bolla Narasimha Rao,c Syed Imthiajali,a T. Lakshminarayan Reddy,b S. N. C. V. L. Pushpavalli,b Nagula Shankaraiah,c Manika Pal-Bhadra,*b
aMedicinal Chemistry & Pharmacology, bChemical Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
cDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India
dCentre for Neuroscience and Cell Biology, Institute for Interdisciplinary Research, University of Coimbra (IIIUC) 3004517, Coimbra, Portugal
Abstract: A series of 4 -[4 -(1-(aryl)ureido)benzamide]podophyllotoxin congeners (11a–
l) were synthesized and evaluated for their cytotoxic activity against six human cancer
cell lines. Some of the compounds like 11a, 11h, 11k and 11l showed significant anti-
proliferative activity in Colo-205 cells and were superior to etoposide. The flow-
cytometric analysis studies indicated that these compounds show strong G1 cell cycle
arrest, as well exhibited improved inhibitory activities on DNA topoisomerase I and II
enzymes. These compounds induce apoptosis by up regulating caspase-3 protein as
observed by ELISA and Western blotting analysis. In addition, a brief structure–activity
relationship studies within the series along with docking results of representative
isobenzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in dry CH2Cl2 (10
mL), 3,5-bis(trifluoromethyl)phenyl isocyanate (g), (115 mg, 0.56 mmol) was added at 0 oC and the stirring was continued for 1 h at the room temperature. After 1 h, the formed
solid was filtered and washed with CH2Cl2 thoroughly to obtain the pure compound 11g
in 220 mg, 77% yield. Mp: 213–215 oC; [α]D25 = – 76.9 (c = 0.5 in CHCl3); 1H NMR
[4',5':4,5]benzo[f]isobenzofuran-5-yl)-4-aminobenzamide (10), (200 mg, 0.37 mmol) in
dry CH2Cl2 (10 mL), 2-chloroethyl isocyanate (k), (0.07 mL, 0.56 mmol) was added at 0 oC and the stirring was continued for another 1 h at the room temperature. After 1 h, the
formed solid was filtered and washed with CH2Cl2 thoroughly to obtain the pure
compound 11k in 160 mg, 66% yield. Mp: 201–203 oC; 1[α]D25 = – 86.7 (c = 0.5 in
The synthesized compounds 11a–l have been evaluated for their in vitro cytotoxicity
in human cancer cell lines. A protocol of 48 h continuous drug exposure has been used
and a sulforhodamine B (SRB) protein assay has been used to estimate cell viability or
growth. The cell lines were grown in DMEM medium containing 10% fetal bovine serum
and 2 mM L-glutamine and were inoculated into 96 well microtiter plates in 90 microliter
at plating densities depending on the doubling time of individual cell lines. The microtiter
plates were incubated at 37 oC, 5% CO2, 95% air, and 100% relative humidity for 24 h
prior to addition of experimental drugs. Aliquots of 10 microliter of the drug dilutions
were added to the appropriate microtiter wells already containing 90 microliter of cells,
sulting in the required final drug concentrations. For each compound four concentrations
(0.1, 1, 10 and 100 µM) were evaluated and done in triplicate wells. Plates were
incubated further for 48 h and assay was terminated by the addition of 50 microliter of
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cold trichloro acetic acid (TCA) (final concentration, 10% TCA) and incubated for 60
min at 4 oC. The plates were washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (50 mL) at 0.4% (w/v) in 1% acetic acid was added to
each of the cells, and plates were incubated for 20 min at room temperature. The residual
dye was removed by washing five times with 1% acetic acid. The plates were air dried.
Bound stain was subsequently eluted with 10 mM trizma base, and the absorbance was
read on an ELISA plate reader at a wavelength of 540 nm with 690 nm reference
wavelengths. Percent growth was calculated on a plate by plate basis for test wells
relative to control wells. The above determinations were repeated three times. Percentage
growth was expressed as the (ratio of average absorbance of the test well to the average
absorbance of the control wells) *100. Growth inhibition of 50% (GI50) was calculated
from [(Ti - Tz)/(C - Tz)] *100 ¼ 50, which is the drug concentration resulting in a 50%
reduction in the net protein increase (as measured by SRB staining) in control cells
during the drug incubation. Where, Tz ¼ Optical density at time zero, OD of control ¼ C,
and OD of test growth in the presence of drug ¼ Ti.
7.2. Cell culture:
Human colon (Colo-205) and breast (MDA-MB-231) carcinoma cells were
purchased from American Type Culture collection Centre (ATCC). Dulbecco’s modified
Eagle’s medium (DMEM) (Invitrogen), supplemented with 2 mM glutamax (Invitrogen),
10% fetal calf serum and 100 U/ml Penicillin and 100 mg/ml streptomycin sulfate
(Sigma) was used as growth media for MDA-MB-231 cells. RPMI medium
supplemented with 10% FCS with appropriate antibiotics (Penicillin, Streptomycin and
Kanamycin) was used for growing Colo-205 cells. These cell lines were maintained at 37 oC in a humidified atmosphere containing 5% CO2 in the incubator.
7.3. MTT assay:
Individual wells of a 96-well tissue culture micro titer plate were inoculated with 100
µL of complete medium containing 1 104 cells. The plates were incubated at 37 °C in a
humidified 5% CO2 incubator for 18 h prior to the experiment. After medium removal,
100 µL of fresh medium containing the test compounds 11a, 11h, 11k, 11l and etoposide
(Eto) at different concentrations such as 0.5, 1, 2 and 4 µM were added to each well and
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incubated at 37 °C for 24 h. Then the medium was discarded and replaced with 10 µL
MTT dye. Plates were incubated at 37 ˚C for 2 h. The resulting formazan crystals were
solubilized in 100 µL extraction buffer. The optical density (O.D) was read at 570 nm
with micro plate reader (Multi-mode Varioskan Instrument-Themo Scientific). The
percentage of DMSO in the medium never exceeded 0.25%.
7.4. Trypan blue exclusion test of cell viability
This method is used to determine the number of viable cells present in cell
suspension. This method is based on the principle that live cells possess intact cell
membranes that excludes trypan blue; whereas dead cells are not capable of excluding
trypan blue. Here viable cells show a clear cytoplasm where as non viable cells show blue
colour cytoplasm. Compounds were treated for 24 h time period. After compound
treatment (11a, 11h, 11k, 11l and etoposide (Eto) at 1, 2 and 4 µM concentrations, the
cells were trypsinized and the dead and viable cells were counted. Each sample was
assayed for triplicates. In this assay 10 µl of 0.4% solution of trypan blue was added to
100 µl of cells. Once after mixing the sample was loaded on to haemocytometer and
examined immediately. The percentage of dead cells was calculated.
7.5. ATP content bioluminescence assay:
The content of intracellular ATP was measured by bioluminescent assay on the basis
of the measurement of the light output of the luciferin-luciferase reaction. The luciferin-
luciferase was purchased as a kit from Promega Company. Colo-205 cells were treated
with concentration of 2 µM for 24 h, then cells were harvested and lysed after treatment
with ice cold RIPA buffer, and cell extracts were obtained. After centrifugation at 12,000
rpm for 2 minutes at 4 oC to remove cell debris, we collected supernatants for ATP
measurement. The amount of ATP was determined by the ATP monitoring kit.
7.6. Cell cycle analysis:
For flow cytometric analysis of DNA content, 5 105 Colo-205 cells in exponential
growth were treated at a concentration of 2 µM of 11a, 11h, 11k, 11l and etoposide (Eto)
for 24 h. After the incubation period, the cells were collected, centrifuged and fixed with
ice-cold 70% ethanol at 4 oC for 30 min. Then the cells were incubated with 1 mg /ml
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RNAase A solution (Sigma) at 37 oC for 30 min followed by staining with 250 µL of
DNA staining solution [10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate, and
0.03 mL of Triton X-100 were dissolved in 100 mL of sterile MilliQ water at room
temperature for 30 min in the dark]. The DNA contents of 20,000 events were measured
by flow cytometer (DAKO CYTOMATION, Beckman Coulter, and Brea, CA).
Histograms were analyzed using Summit Software.
7.7. DNA topoisomerase-IIα inhibition assay:
The mixture of 200 ng of super coiled pBR322 plasmid DNA and 4 units of human
DNA topoisomerase-IIα (Sigma, USA) was incubated with the compounds [11a-11l and
etoposide (Eto)] in the assay buffer (10 mM Tris–HCl (pH 7.9) containing 50 mM NaCl,
5 mM MgCl2, 1 mM EDTA, 1 mM ATP, and 15 µg/mL bovine serum albumin) for 30
min at 30 oC. The reaction in a final volume of 20 µL was terminated by the addition of 3
µL of 7 mM EDTA. Reaction products were analyzed on a 1% Agarose gel at 60V for 1
h with a running buffer of TAE. Gels were stained for 30 min in an aqueous solution of
ethidium bromide (0.5 µg/mL). DNA bands were visualized by transillumination with
UV light.
7.8. DNA topoisomerase-I inhibition assay:
The activity of DNA topoisomerase-I was determined by assessing the relaxation of
supercoiled DNA pBR322. The mixture of 500 ng of plasmid pBR322 DNA and 0.4 units
of recombinant human DNA topoisomerase-I (TopoGEN INC., USA) was incubated
without and with the synthesized compounds (11a-11l) at 37 0C for 30 min in the
relaxation buffer (10 mM Tris–HCl (pH 7.9), 150 mM NaCl, 0.1% bovine serum
albumin, 1 mM spermidine, 5% glycerol). The reaction in the final volume of 10 µL was
terminated by adding 2.5µ L of the stop solution containing 5% sarcosyl, 0.0025%
bromophenol blue, and 25% glycerol. DNA samples were then electrophoresed on a 1%
agarose gel.
7.9. Caspase-3 assay:
The caspase-3 fluorescent assay kit (Clonetech, CA) was applied to evaluate the
caspase-3 activity, using the procedures provided by the manufacturer. Colo-205 cells
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were treated with compounds 11a, 11h, 11k, 11l and etoposide (Eto) at 2 µM
concentration for 24h. Cell lysates were added to the 2X reaction buffer containing DTT
and caspase substrate was added and incubation was carried out at 37 C for 1 h.
Readings were taken at excitation wavelength 400 nm and emission wavelength 505 nm.
7.10. Protein extraction and Western blot analysis:
Colo-205 human colon cancer cells were seeded in 60 mm dishes and were allowed to
grow to attain 80% confluency for 24 h. Compounds (11a, 11h, 11k, 11l and etoposide
(Eto)) were added to the culture media for a final concentration of 2 µM, and the cells
were incubated with compounds for 24 h. After 24 h, total cell lysates from cultured colo-
205 cells were obtained by lysing the cells in ice-cold RIPA buffer (1X PBS, 1% NP- 40,
a50 Growth inhibition and the values are mean of three determinations; bbreast cancer, coral cancer, dcolon cancer, elung cancer, fcervix cancer, govarian cancer, hEtoposide, iADR (adriamycin), NA = not active
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Figure 2A. Colo-205 and MDA-MB-231 cells were treated with compounds 11a, 11h,
11k, 11l and etoposide (Eto) at 0.5, 1, 2 and 4 µM concentration for 24 h. The MTT cell
viability assay was conducted and the optical density (O.D) was observed at 570 nm.
DMSO indicates the untreated cells.
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Figure 2B. The number of viable cells present after 24 h was measured in compound
treated [11a, 11h, 11k, 11l and etoposide (Eto)] Colo-205 cells.
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Figure 2C. The effect of compounds on cellular energy (ATP) in Colo-205 cells was
measured by luminescence based assay. Compounds at 2 µM concentration have strongly
depleted the cellular ATP. The error bar in the graph represents the standard deviation
(S.D) obtained from three independent experiments. ***indicate P<0.001. Con:
represents control untreated cells.
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Figure 3. Colo-205 cells were treated with compounds 11a, 11h, 11k, 11l and etoposide
(Eto) at 2 µM concentration for 24 h. The cells were further processed for FACS
analysis. Compounds 11a, 11h, 11k and 11l have shown G1 cell cycle arrest. Control
4 -[4 -(1-(Aryl)ureido)benzamide]podophyllotoxins as DNA Topoisomerase I and IIα Inhibitors and Apoptosis Inducing Agents
Ahmed Kamal,*a Paidakula Suresh,a M. Janaki Ramaiah,b T. Srinivasa Reddy,a,c Ravi Kumar Kapavarapu,d Bolla Narasmha Rao,c Syed Imthiajali,a T. Lakshminarayan Reddy,b
S. N. C. V. L. Pushpavalli,b Nagula Shankaraiah,c Manika Pal-Bhadra,*b
A series of new 4 -[4 -(1-(aryl)urea)benzamido]podophyllotoxin congeners (11a–l) were
synthesized and evaluated for their in vitro anticancer activity against six human cancer
cell lines. Among them, compounds 11a, 11h, 11k and 11l showed significant inhibitory
activities on DNA topoisomerase I and II , strong G1 cell cycle arrest and induces
apoptosis, and these results are supported by molecular docking studies.